Psoriasis upregulated phorbolin-1 shares structural but not functional similarity to the mRNA-editing protein apobec-1.

Earlier studies of psoriatic and normal primary keratinocytes treated with phorbol 12-myristate-1-acetate identified two low-molecular-weight proteins, termed phorbolin-1 (20 kDa; pI 6.6) and phorbolin-2 (17.6 kDa; pI 6.5). As a first step towards elucidating the role of these proteins in psoriasis, we report here the molecular cloning and chromosomal mapping of phorbolin-1 and a related cDNA that codes for a protein exhibiting a similar amino acid sequence. The phorbolins were mapped to position 22q13 immediately centromeric to the c-sis proto-oncogene. Transient expression of the phorbolin-1 cDNA in COS cells and by in vitro transcription/translation, yielded polypeptides that comigrated with phorbolins-1 and -2. Comparative sequence analysis revealed 22% overall identity and a similarity of 44% of the phorbolins to apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme; however, recombinant-expressed phorbolin-1 exhibited no cytidine deaminase activity, using either a monomeric nucleoside or apolipoprotein B cRNA as substrate, and failed to bind an AU-rich RNA template. Whereas the precise function of the phorbolins remains to be elucidated, the current data suggest that it is unlikely to include a role in the post-transcriptional modification of RNA in a manner analogous to that described for apobec-1.

A cytidine deaminase expressed in the post-infective L3 stage of the filarial nematode, Brugia pahangi, has a novel RNA-binding activity.

A number of genes have been identified that are highly expressed in the post-infective L3 stage of the filarial parasite, Brugia pahangi. Amongst these was a cDNA with homology to the cytidine deaminase (CDD) gene family. Phylogenetic analysis of the various cytosine nucleoside deaminases suggest that Brugia pahangi CDD evolved with significant divergence from the RNA editing family. In order to characterize its function, we have expressed Brugia pahangi CDD in bacteria as a chimera with maltose-binding protein (MBP). Biochemical analysis demonstrates the MBP-CDD fusion protein functions as an authentic cytidine deaminase with an obligate requirement for zinc. In addition to cytidine deaminase activity, however, the fusion protein demonstrates RNA binding activity with specificity for AU-rich sequences and was found to bind an RNA template spanning the edited site of mammalian apolipoprotein B (apoB) mRNA. This RNA binding activity was not found in two different recombinant bacterial CDD proteins. In vitro RNA editing assays revealed that MBP-CDD failed to mediate cytidine deamination of a mammalian apoB RNA template. Furthermore, binding of MBP-CDD to the apoB RNA did not inhibit in vitro editing of this template by apobec-1. The data suggest that the cytosine nucleoside deaminases and RNA editing deaminases have acquired different mechanisms of binding to an AU-rich RNA template, presumably with different functional implications.

apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme, is a novel RNA-binding protein.

Apolipoprotein B (apoB) mRNA editing is mediated by an enzyme complex which includes the catalytic subunit, apobec-1. Recombinant GST/APOBEC-1 binds with high specificity to a rat apoB RNA template as demonstrated by UV cross-linking and electrophoretic mobility shift assay (EMSA). ApoB RNA binding was competed by poly(U), poly(A,U), and tRNA, but not by poly(A) or other homopolymeric ribonucleotides. UV cross-linking of GST/APOBEC-1 to an apoB RNA template was uninfluenced by the binding of proteins of approximately 60 and approximately 44 kDa, present in S100 extracts prepared from different sources. The binding of these proteins was similarly uninfluenced by the simultaneous binding of GST/APO-BEC-1. Moreover, the inclusion of heterologous S100 extracts in the RNA binding reactions completely abrogated the competitive displacement of GST/APOBEC-1 by tRNA. EMSA revealed the onset of RNA binding within 1-2 min, and its specificity was confirmed by a supershift with anti-GST/APOBEC-1 antisera. The structural specificity for apoB RNA binding, as inferred from EMSA, appears to be distinct from apoB RNA editing since wild-type chicken apoB RNA, which is not editable, and several mutant chicken apoB RNAs containing clustered mutations within the minimal apoB RNA editing cassette, bound with efficiency similar to the rat apoB RNA template. In conclusion, while the data suggest that apobec-1 binds AU-rich templates, the importance of this observation in the context of mammalian apoB mRNA editing remains unknown.

Mutagenesis of apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme, reveals distinct domains that mediate cytosine nucleoside deaminase, RNA binding, and RNA editing activity.

Apolipoprotein (apo) B48 is synthesized by mammalian small intestine as a result of post-transcriptional RNA editing. This process is mediated by an enzyme complex containing a catalytic subunit, apobec-1, which is homologous to other cytidine deaminases, particularly in a domain (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C which coordinates zinc, apobec-1, expressed as a glutathione S-transferase fusion protein, demonstrates both apoB RNA editing and cytidine deaminase activity. His61, Cys93, and Cys96, the putative zinc-coordinating residues, were mutated to Arg, Ser, and Ser, respectively, with loss of RNA editing activity and either great reduction or abolition of cytidine deaminase activity. Mutation of the catalytically active Glu63 residue to Gln and Pro92 to Leu abolished both cytidine deaminase and RNA editing activity. The conservative His61-->Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity. Thus, zinc binding is required for both apoB RNA editing and cytidine deaminase activity. Mutation of the first four leucines within the heptad repeat of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity. GST/APOBEC-1 was also demonstrated to cross-link to apoB RNA. Mutation of His61-->Arg abolished RNA binding, while the Glu63-->Gln and Cys96-->Ser mutant proteins showed wild-type levels of RNA binding. The remaining mutants had reduced levels of activity. Overexpression of wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB. Transfection of the His61-->Cys, LRR, and Cys93-->Ser mutants increased endogenous editing 2-3-fold, while Glu63-->Gln and His61-->Arg mutants acted as dominant negatives, reducing endogenous editing. These data suggest that apobec-1 has distinct functional domains which modulate activity in the context of the apoB mRNA editing enzyme.

Apolipoprotein B messenger RNA editing: insights into the molecular regulation of post-transcriptional cytidine deamination.

A site-specific cytidine deamination (cytidine to uridine) in nuclear apolipoprotein B messenger RNA creates a translational stop codon that produces apolipoprotein B48. This process is mediated by an enzyme composed of distinct subunits, including apolipoprotein B messenger RNA editing enzyme catalytic polypeptide-1 and additional complementation factors. The apolipoprotein B messenger RNA editing enzyme catalytic polypeptide-1 is expressed ubiquitously in the rat, but is largely confined to the small intestine in humans and rabbits. By contrast, complementation activity is present in tissues that neither express nor edit apolipoprotein B messenger RNA.

The binding of apobec-1 to mammalian apo B RNA is stabilized by the presence of complementation factors which are required for post-transcriptional editing.

C to U RNA editing in mammalian intestinal apolipoprotein B mRNA creates an in-frame translational stop and the synthesis of a truncated protein called apo B48. This site specific cytidine deamination is mediated by an enzyme complex of which the catalytic component (apobec-1) is a 27 kDa zinc-binding protein. apobec-1, expressed in bacteria, will bind to mammalian apo B RNA as well as a number of other AU-rich RNA templates. Apo B RNA-binding activity can be competed by the addition of tRNA, an effect which can be overcome by the addition of complementation factors such as chick enterocyte S-100 extracts. Thus, apobec-1 may be a non-specific RNA binding protein which requires the presence of complementation factors to stabilize and enhance its binding in the setting of the holo-enzyme.